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Abstract:

A method of modulating transplant organ size in a subject in need thereof
is disclosed. The method comprising: (a) administering to the subject an
agent capable of modulating an activity or expression of a coagulation
factor or an effector thereof; and (b) transplanting the organ into the
subject; thereby modulating the transplant organ size in the subject.

Claims:

1. A method of modulating transplant organ size in a subject in need
thereof, the method comprising: (a) administering to the subject an agent
capable of modulating an activity or expression of a coagulation factor
or an effector thereof; and (b) transplanting the organ into the subject;
thereby modulating the transplant organ size in the subject.

2-5. (canceled)

6. An article of manufacture comprising a packaging material packaging an
immunosuppressing agent and an agent capable of modulating an activity or
expression of a coagulation factor or an effector thereof.

23. The method of claim 22, wherein said agent is capable of
up-regulating said activity or expression of said coagulation factor or
an effector thereof.

24-40. (canceled)

41. The method of claim 1, wherein said subject is a human being.

42-44. (canceled)

45. The method of claim 1, wherein said modulating an activity or
expression of a coagulation factor or an effector thereof is effected
prior to, concomitantly with or following transplantation.

46. The method of claim 1, further comprising conditioning the subject
prior to transplanting so as to prevent organ rejection.

Description:

FIELD AND BACKGROUND OF THE INVENTION

[0001] The present invention, in some embodiments thereof, relates to
coagulation factors and effectors of same, and more particularly, but not
exclusively, to the modulation of same for control of transplant organ
size.

[0002] Organ transplants are commonly used for treatment of organ failure,
however, there is a major shortage in donor organs and the difference
between supply and demand continues to grow every year. Transplantation
of organs including kidney, heart, liver, lung and pancreas, are carried
out following assessment of several factors including organ size, blood
type, tissue type, medical urgency of the subject's illness and time
already spent on the waiting list. The organ is offered first to the
candidate who is the best match in all the above criteria, but
nevertheless, recipients usually wait for prolonged periods of time
before a transplant can be found and often no matched transplants are
found resulting in the death of many patients every year.

[0003] Despite great progress in molecular genetics and embryogenesis,
size control of tissues and organs remains a mystery. Organ size control
during embryonic development or in tissue regeneration, involves a fine
balance between cell growth, proliferation and death, maintained by
extrinsic and intrinsic factors. Even though the size of an organ or
organism depends largely on cell numbers and cell size, studies have
found that the simple deregulation of cell proliferation or cell growth
does not necessarily lead to changes in organ size. Some important
insights into organ size control were provided in the past few decades
from studies of the Drosophila imaginal disc model. In general, it seems
that extrinsic mechanisms are associated with nutrition or systemic
growth factors operating through the Insulin/PI3K and the TOR pathways,
while intrinsic mechanisms are likely linked to patterning morphogenes
and apoptosis-signaling complexes. Overall, experimental data suggest
that organ size might be regulated by a `total mass checkpoint` mechanism
which functions to link the regulation of cell size and cell
proliferation [Christopher et al., Curr Opin in Genet Develop (2001)
11(3) 279-286].

[0004] Recent evidence suggests that in some organs, such as the pancreas,
organ size is intrinsically defined by the size of the stem cell pool
committed to the development of the organ. Thus, using genetic
manipulations leading to reduction of the size of the stem cell pool it
was demonstrated that a smaller pancreas was generated in animals with
reduced stem cells [Stanger et al., Nature (2007) 445: 886-91], while in
contrast, similar genetic manipulations of liver stem cells did not
affect the ability of the liver to regenerate and regain its normal size
[Stanger et al., supra]. This difference might indicate that autonomous
growth of the embryonic pancreas tissue exhibits total dependence on
intrinsic elements, namely the size of the stem cell pool, in contrast to
the embryonic liver, which is likely controlled by additional extrinsic
factors. These recent observations are reminiscent of the early studies
of Metcalf who showed that following transplantation of several pieces of
embryonic thymus, each one attained the full size of an adult thymus
[Metcalf D., Aust J Exp Biol Med Sci (1963) 41, SUPPL437-47], while
transplanting embryonic spleen tissue, the combined size of all the
spleen implants was similar in size to a single adult spleen [Metcalf D.,
Transplantation (1964) 2: 387-92].

[0005] As stated above, it is generally accepted that liver regeneration
is not dependent on progenitor cells or stem cells but rather on
extrinsic factors. According to the teachings of Michalopoulos and
DeFrances [Michalopoulos and DeFrances, Science (1997) 276: 60-66]
transplanting livers from large dogs into small dogs results in a gradual
decrease in liver size until the size of the organ becomes proportional
to the new body size. On the contrary, when baboon livers are
transplanted into humans, the transplanted intact livers of baboon origin
rapidly grow in size (within a week) until reaching the size of a human
liver. These results demonstrate that liver mass is regulated and that
signals from the body can have negative as well as positive effects on
liver mass until the correct size is reached. Furthermore, according to
their teachings, liver regeneration is an orchestrated response induced
by specific external stimuli and involving sequential changes in gene
expression, growth factor production, and morphologic structure. Many
growth factors and cytokines, most notably hepatocyte growth factor,
epidermal growth factor, transforming growth factor-α,
interleukin-6, tumor necrosis factor-α, insulin and norepinephrine,
appear to play an important role in liver regeneration.

SUMMARY OF THE INVENTION

[0006] According to an aspect of some embodiments of the present invention
there is provided a method of modulating transplant organ size in a
subject in need thereof, the method comprising: (a) administering to the
subject an agent capable of modulating an activity or expression of a
coagulation factor or an effector thereof; and (b) transplanting the
organ into the subject; thereby modulating the transplant organ size in
the subject.

[0007] According to an aspect of some embodiments of the present invention
there is provided a use of an agent capable of down-regulating an
activity or expression of a coagulation factor or an effector thereof for
enhancing a transplant organ size in a subject.

[0008] According to an aspect of some embodiments of the present invention
there is provided a use of an agent capable of up-regulating an activity
or expression of a coagulation factor or an effector thereof for
decreasing a transplant organ size in a subject.

[0009] According to an aspect of some embodiments of the present invention
there is provided a pharmaceutical composition comprising an agent
capable of down-regulating an activity or expression of a coagulation
factor or an effector thereof for enhancing a transplant organ size in a
subject.

[0010] According to an aspect of some embodiments of the present invention
there is provided a pharmaceutical composition comprising an agent
capable of up-regulating an activity or expression of a coagulation
factor or an effector thereof for decreasing a transplant organ size in a
subject.

[0011] According to an aspect of some embodiments of the present invention
there is provided an article of manufacture comprising a packaging
material packaging an immunosuppressing agent and an agent capable of
modulating an activity or expression of a coagulation factor or an
effector thereof.

[0012] According to some embodiments of the invention, the modulating
transplant organ size comprises enhancing the transplant organ size.

[0013] According to some embodiments of the invention, the agent is
capable of down-regulating the activity or expression of the coagulation
factor or an effector thereof.

[0014] According to some embodiments of the invention, the coagulation
factor or an effector thereof is selected from the group consisting of
Factor VIII, Factor X, Factor Xa, Prothrombin, Thrombin, Factor XIII,
Factor XIIIa and PAR.

[0015] According to some embodiments of the invention, the agent is
capable of down-regulating an activity or expression of Factor VIII in
the subject.

[0016] According to some embodiments of the invention, the agent is
capable of down-regulating an activity or expression of Factor Xa in the
subject.

[0017] According to some embodiments of the invention, the agent is
Clexane.

[0018] According to some embodiments of the invention, the agent is
capable of down-regulating an activity or expression of Thrombin in the
subject.

[0019] According to some embodiments of the invention, the agent is
selected from the group consisting of Clexane and Dabigatran.

[0020] According to some embodiments of the invention, the agent is
capable of up-regulating an activity or expression of antithrombin in the
subject.

[0021] According to some embodiments of the invention, the agent is
capable of down-regulating an activity or expression of PAR1 in the
subject.

[0022] According to some embodiments of the invention, the agent is as set
forth in SEQ ID NO: 15.

[0023] According to some embodiments of the invention, the agent is
capable of down-regulating an activity or expression of PAR4 in the
subject.

[0024] According to some embodiments of the invention, the agent is as set
forth in SEQ ID NO: 16.

[0025] According to some embodiments of the invention, the agent further
comprises G-CSF.

[0026] According to some embodiments of the invention, the agent is an
oligonucleotide silencing agent.

[0027] According to some embodiments of the invention, the modulating
transplant organ size comprises decreasing the organ size.

[0028] According to some embodiments of the invention, the agent is
capable of up-regulating the activity or expression of the coagulation
factor or an effector thereof.

[0029] According to some embodiments of the invention, the coagulation
factor or an effector thereof is selected from the group consisting of
Factor VIII, Factor X, Factor Xa, Prothrombin, Thrombin, Factor XIII,
Factor XIIIa and PAR.

[0030] According to some embodiments of the invention, the agent is
capable of up-regulating an activity or expression of Factor VIII in the
subject.

[0031] According to some embodiments of the invention, the agent is
capable of up-regulating an activity or expression of Thrombin in the
subject.

[0032] According to some embodiments of the invention, the agent is
capable of down-regulating an activity or expression of antithrombin in
the subject.

[0034] According to some embodiments of the invention, the organ comprises
a solid tissue.

[0035] According to some embodiments of the invention, the organ comprises
a liver.

[0036] According to some embodiments of the invention, the organ comprises
a spleen.

[0037] According to some embodiments of the invention, the organ comprises
a pancreas.

[0038] According to some embodiments of the invention, the organ is
derived from a prenatal organism.

[0039] According to some embodiments of the invention, the organ is
derived from a post natal organism.

[0040] According to some embodiments of the invention, the organ is
derived from an adult.

[0041] According to some embodiments of the invention, the organ is
derived from a xenogeneic donor.

[0042] According to some embodiments of the invention, the xenogeneic
donor is a pig.

[0043] According to some embodiments of the invention, the organ is
derived from an allogeneic donor.

[0044] According to some embodiments of the invention, the organ is
derived from a syngeneic donor.

[0045] According to some embodiments of the invention, the organ is
derived from a cadaver donor.

[0046] According to some embodiments of the invention, the subject is a
human being.

[0047] According to some embodiments of the invention, the subject in need
thereof has a hepatic disorder.

[0048] According to some embodiments of the invention, the subject in need
thereof has a renal disorder.

[0049] According to some embodiments of the invention, the subject in need
thereof has a pancreatic disorder.

[0050] According to some embodiments of the invention, the modulating an
activity or expression of a coagulation factor or an effector thereof is
effected prior to, concomitantly with or following transplantation.

[0051] According to some embodiments of the invention, the method further
comprising conditioning the subject prior to transplanting so as to
prevent organ rejection.

[0052] Unless otherwise defined, all technical and/or scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention pertains. Although
methods and materials similar or equivalent to those described herein can
be used in the practice or testing of embodiments of the invention,
exemplary methods and/or materials are described below. In case of
conflict, the patent specification, including definitions, will control.
In addition, the materials, methods, and examples are illustrative only
and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Some embodiments of the invention are herein described, by way of
example only, with reference to the accompanying drawings. With specific
reference now to the drawings in detail, it is stressed that the
particulars shown are by way of example and for purposes of illustrative
discussion of embodiments of the invention. In this regard, the
description taken with the drawings makes apparent to those skilled in
the art how embodiments of the invention may be practiced.

[0068] The present invention, in some embodiments thereof, relates to
coagulation factors and effectors of same, and more particularly, but not
exclusively, to the modulation of same for control of transplant organ
size.

[0069] The principles and operation of the present invention may be better
understood with reference to the drawings and accompanying descriptions.

[0070] Before explaining at least one embodiment of the invention in
detail, it is to be understood that the invention is not necessarily
limited in its application to the details set forth in the following
description or exemplified by the Examples. The invention is capable of
other embodiments or of being practiced or carried out in various ways.
Also, it is to be understood that the phraseology and terminology
employed herein is for the purpose of description and should not be
regarded as limiting.

[0071] While reducing the present invention to practice, the present
inventors have surprisingly uncovered that various factors in the blood
coagulation pathway play a key role in modulating transplant organ size
without compromising tissue functionality. Apparently, but without being
bound by theory, coagulation factors or absence thereof mediates this
novel activity by modulating key genes known to participate in
coagulation, endothelium formation, patterning and differentiation.

[0073] Moreover, it is shown herein that Factor VIII is involved in
regulation of organ size in situations in which there is a drive for
oversized growth. For example, treatment of hemophilic mice with G-CSF
lead to splenomegaly in these mice (FIG. 5A-B). As depicted in FIGS.
4A-B, embryonic implants of different sources (e.g. pig, mouse) are
endowed with stem cell pools of different sizes prior to transplantation.
These tissues are therefore likely to exhibit different organ size upon
completion of growth and differentiation. As a result of Factor VIII
depletion, the hemophilic mice lacking the potential inhibitory activity
(i.e. overgrowth checkpoint) exhibit much larger pig transplanted organ
sizes (FIG. 4C). Similarly, down regulation of other coagulant factors,
such as Factor Xa and Thrombin, had major influence on oversized organs
(FIGS. 8B-C and 9). Taken together, all these findings substantiate the
use of coagulation factors or effectors thereof for the modulation of
transplant organ size according to the source of the organ and the
intended use.

[0074] Thus, according to one aspect of the present invention there is
provided a method of modulating transplant organ size in a subject in
need thereof. The method comprising: administering to the subject an
agent capable of modulating an activity or expression of a coagulation
factor or an effector thereof; and transplanting the organ into the
subject; thereby modulating the transplant organ size in the subject.

[0075] As used herein, the term "modulating" refers to a change in size of
the transplanted organ in the host, either an increase (e.g., at least
5%, 10%, 15%, 20%, 30%, 50%, 100%, 200%, 250%, 400% or more) or a
decrease (e.g., at least 5%, 10%, 15%, 20%, 30%, 50%, 100%, 200%, 250%,
400% or more). Modulation is typically determined with respect to an
untreated subject (i.e., who was not subject to modulation of a
coagulation factor or an effector thereof). Modulation can be determined
by any method known to one of ordinary skill in the art, as for example
by activity assays such as measurement of blood insulin or albumin levels
for determination of pancreas or liver transplant organ sizes,
respectively, or by using any suitable, widely practiced, imaging methods
including computerized tomography (CT) and ultrasound imaging. If a
plurality of observations are made, one skilled in the art can apply any
routine statistical analysis to identify such modulations. Typically,
according to some embodiments of the present invention, modulating
transplant organ size is not accompanied by changes in functionality of
the transplanted tissue.

[0076] As used herein, the phrase "subject in need thereof" refers to a
mammal, preferably a human being, male or female at any age that is in
need of organ transplantation. Typically the subject is in need of organ
transplantation (also referred to herein as recipient) due to a disorder
or a pathological or undesired condition, state, or syndrome, or a
physical, morphological or physiological abnormality which is amenable to
treatment via organ transplantation. Examples of such disorders are
provided further below. Moreover, the subject is typically not diagnosed
with a coagulation factor disorder.

[0077] As used herein, the term "organ" refers to a bodily tissue which
may be transplanted in full or in part, including solid tissues and soft
tissues. Exemplary organs which may be transplanted according to the
present teachings include, but are not limited to, liver, pancreas,
spleen, kidney, heart, lung, skin, intestine and lymphoid/hematopoietic
tissues (e.g. lymph node, Peyer's patches thymus or bone marrow). It will
be appreciated that the organ of the present invention is not an embryo
or fetus.

[0078] As used herein, the phrase "transplant organ size" refers to the
size of an organ transplanted from one body to another. The transplant
organ size may be evaluated in comparison to the average size of an
identical organ transplanted to a host of the same species, age group,
medical condition and gender as the subject. Transplant organ size is
evaluated post transplantation and optionally prior to the
transplantation.

[0079] Transplanting the organ may be effected in numerous ways, depending
on various parameters, such as, for example, the graft type; the type,
stage or severity of the recipient's organ failure; the physical or
physiological parameters specific to the subject; and/or the desired
therapeutic outcome. Depending on the application and purpose,
transplanting the organ may be effected using an organ originating from
any of various mammalian species, by implanting the organ into various
anatomical locations of the subject, using an organ consisting of a whole
or partial organ or tissue, and/or by using a transplant consisting of
various numbers of discrete organs, tissues, and/or portions thereof.

[0080] Optionally, when transplanting an organ of the present invention
into a subject having a defective organ, it may be advantageous to first
at least partially remove the failed organ from the subject so as to
enable optimal development of the transplant, and structural/functional
integration thereof with the anatomy/physiology of the subject.

[0081] Depending on the application, the method may be effected using an
organ which is syngeneic or non-syngeneic with the subject.

[0082] As used herein, an organ which is "syngeneic" with the subject
refers to an organ which is derived from an individual who is essentially
genetically identical with the subject. Typically, essentially fully
inbred mammals, mammalian clones, or homozygotic twin mammals are
syngeneic.

[0083] Examples of syngeneic organs include an organ derived from the
subject (also referred to in the art as an "autologous organ"), a clone
of the subject, or a homozygotic twin of the subject.

[0084] As used herein, an organ which is "non-syngeneic" with the subject
refers to an organ which is derived from an individual who is allogeneic
or xenogeneic with the subject's lymphocytes.

[0085] As used herein, an organ which is "allogeneic" with the subject
refers to an organ which is derived from a donor who is of the same
species as the subject, but which is substantially non-clonal with the
subject. Typically, outbred, non-zygotic twin mammals of the same species
are allogeneic with each other.

[0086] As used herein, an organ which is "xenogeneic" with the subject
refers to an organ which substantially expresses antigens of a different
species relative to the species of a substantial proportion of the
lymphocytes of the subject. Typically, outbred mammals of different
species are xenogeneic with each other.

[0087] As is described and illustrated in the Examples section below,
porcine organs were transplanted into immunodeficient mice which were
hemophilic or non-hemophilic. These organs developed into well developed
and tolerated functional organs of porcine lineage.

[0088] Porcine organs are widely considered to be a potentially ideal
animal alternative to human organs for therapeutic transplantation in
humans due to their morphological compatibility with the human anatomy,
and due to their essentially unlimited supply which would overcome the
restricted availability impediment inherent to prior art human organs
[Auchincloss, H. and Sachs, D. H., Annu. Rev. Immunol. (1998) 16,
433-470; Hammerman, M. R., Curr. Opin. Nephrol. Hypertens. (2002) 11,
11-16].

[0089] Organs of porcine origin are preferably obtained from a source
which is known to be free of porcine zoonoses, such as porcine endogenous
retroviruses. Similarly, human-derived organs are preferably obtained
from substantially pathogen-free sources.

[0090] Depending on the application and available sources, the organ may
be obtained from a prenatal organism, postnatal organism, an adult or a
cadaver donor.

[0091] An organ derived from a prenatal organism may be obtained from a
fetus at any gestational stage of pregnancy. It will be understood by one
skilled in the art that a period of gestation corresponds to a
time-period elapsed since fertilization of a developing embryo or fetus.
Thus, the stage of differentiation of a developing organ corresponds to
the developmental stage of the embryo or fetus from which it is derived.
Porcine and human gestational development have been extensively studied
and characterized, and, as such, the ordinarily skilled artisan will
possess the necessary expertise for suitably obtaining a porcine or human
organ at a specific gestational stage so as to enable the practicing of
the present invention.

[0092] Thus, according to an exemplary embodiment, the organ is obtained
from a fetus at a gestational stage which enables optimal organ
functionality and immuno-compatibility without teratoma formation. WO
2003/022123, WO 2004/078022, WO 2006/038211, WO 2006/077592 provide
sufficient guidance for selecting the appropriate gestational stage for
complying with these pre-requisites, each of which is hereby incorporated
by reference in its entirety. For example, as described in the Examples
section which follows, when using a porcine hepatic graft for practicing
hepatic transplantation method of the present invention, the graft is
derived from a porcine liver which may be at a developmental stage
selected from a range of 25 to 56 days of gestation, at a developmental
stage selected from a range of 26 to 56, at a developmental stage
selected from a range of 27 to 56 days of gestation, at a developmental
stage selected from a range of 28 to 56 days of gestation, at a
developmental stage selected from a range of 28 to 42 days of gestation,
at a developmental stage selected from a range of 27 to 29 days of
gestation, or at a developmental stage of 28 days of gestation.

[0093] When using a porcine pancreatic tissue of the present invention,
the graft is derived from a porcine pancreas which may be at a
developmental stage selected from a range of about 42 to about 80 days of
gestation, at a gestational stage of about 42 to about 56 days of
gestation, or at a developmental stage of 42 days of gestation.

[0094] Likewise, when using a porcine splenic tissue of the present
invention, the graft is derived from a porcine spleen which is at a
developmental stage selected from a range of about 42 to about 80 days of
gestation, at a gestational stage of about 42 to about 56 days of
gestation, or at a developmental stage of 42 days of gestation.

[0095] According to an exemplary embodiment of the present invention, the
transplanted organ is obtained from a human being, including a human
fetus. For example, when using a human hepatic tissue, the organ is
preferably derived from a human liver which is at a developmental stage
selected from a range of 6 to 14 weeks of gestation, 6 to 13 weeks of
gestation, 6 to 12 weeks of gestation, 6 to 11 weeks of gestation, 6 to
10 weeks of gestation, 6 to 9 weeks of gestation, 6 to 8 weeks of
gestation, or 7 weeks of gestation.

[0096] The following table provides examples of the gestational stages of
human and porcine grafts at which these can provide grafts which are
essentially at corresponding developmental stages:

TABLE-US-00001
TABLE 1
Corresponding gestational stages of pigs and humans:
Gestational stage Gestational stage
of porcine graft (days) of human graft (days)
18 44
20 49
22 54
23 56-57
25 61-62
26 63
28 68-69
31 75
38 92
42 102
46 112
49 119
56 136
62 151
72 175
80 195
88 214
The gestational stage (in days) of a graft belonging to a given species
which is at a developmental stage essentially corresponding to that of a
porcine graft can be calculated according to the following formula:
[gestational stage of porcine graft in days]/[gestational period of pig
in days] × [gestational stage of graft of given species in days].
Similarly, the gestational stage (in days) of a graft belonging to a
given species which is at a developmental stage essentially corresponding
to that of a human graft can be calculated according to the following
formula: [gestational stage of human graft in days]/[gestational period
of humans in days] × [gestational stage of graft of given species
in days]. The gestational stage of pigs is about 115 days and that of
humans is about 280 days.

[0097] The present invention envisages that organs for transplantation are
derived from species other than human or pig which are at stages of
differentiation corresponding to the presently disclosed optimal
gestational stages. Animals such as the major domesticated or livestock
animals, and primates, which have been extensively characterized with
respect to correlation of stage of differentiation with gestational stage
may be suitable for practicing the present methods. Such animals include
various mammalian species, such as, but are not limited to, bovines
(e.g., cow), equids (e.g., horse), porcines (e.g. pig), ovids (e.g.,
goat, sheep), felines (e.g., Felis domestica), canines (e.g., Canis
domestica), rodents (e.g., mouse, rat, rabbit, guinea pig, gerbil,
hamster), and primates (e.g., chimpanzee, rhesus monkey, macaque monkey,
marmoset) or human beings.

[0098] It will be appreciated that the organ according to the present
invention may also be obtained from a postnatal organism. Thus, the organ
may be obtained from an organism during the period beginning immediately
after birth and extending for about six weeks.

[0099] Furthermore, according to the present teachings, the organ may be
obtained from an adult, either a living or cadaver donor. If the organ is
obtained from a cadaver donor, it is best to obtain the organ within
36-50 hours of death as to enable optimal chances of engraftment and
functionality. In order to minimize rejection of transplanted organs, it
will be appreciated that factors such as blood type and tissue type
should be considered prior to transplantation.

[0100] Various common art methods may be employed to obtain an organ for
transplantation.

[0101] Transplanting an organ of the present invention may be effected by
transplanting the organ into any one of various anatomical locations,
depending on the application. The organ may be transplanted into a
homotopic anatomical location (a normal anatomical location for the organ
transplant), or into an ectopic anatomical location (an abnormal
anatomical location for the transplant). Depending on the application,
the graft may be advantageously implanted under the renal capsule, or
into the kidney, the testicular fat, the sub cutis, the omentum, the
portal vein, the liver, the spleen, the heart cavity, the heart, the
chest cavity, the lung, the pancreas and/or the intra abdominal space.

[0102] For example, a liver of the present invention may be transplanted
into the liver, the portal vein, the renal capsule, the sub-cutis, the
omentum, the spleen, and the intra-abdominal space. Transplantation of a
liver into various anatomical locations such as these is commonly
practiced in the art to treat diseases amenable to treatment via hepatic
transplantation. Similarly, transplanting the pancreas of the present
invention may be advantageously effected by transplanting the tissue into
the portal vein, the liver, the pancreas, the testicular fat, the
sub-cutis, the omentum, an intestinal loop (the subserosa of a U loop of
the small intestine) and/or the intra-abdominal space.

[0103] Following transplantation of the organ into a subject according to
the present teachings, it is advisable, according to standard medical
practice, to monitor the growth functionality and immuno-compatability of
the organ according to any one of various standard art techniques. For
example, as described in the Example section below, the functionality of
a pancreas transplant may be monitored following transplantation by
standard pancreas function tests (e.g. analysis of serum levels of
insulin) Likewise, liver transplant of the present invention may be
monitored following transplantation by standard liver function tests
(e.g. analysis of serum levels of albumin, total protein, ALT, AST, and
bilirubin, and analysis of blood-clotting time). Structural development
of the organ may be monitored via computerized tomography, or ultrasound
imaging.

[0104] Depending on the transplantation context, in order to facilitate
engraftment of the organ, the method may further advantageously comprise
conditioning the subject with an immunosuppressive regimen prior to,
concomitantly with, or following transplantation of the organ.

[0109] It will be appreciated that the inventors of the present invention
have uncovered a direct correlation between Factor VIII expression and
transplant organ size. As clearly depicted in the Examples section
hereinbelow, transplantation of porcine tissue (including spleen,
pancreas and liver) into Factor VIII knock out mice leads to enlarged
organ size compared to wild-type mice without effecting organ
functionality.

[0110] It will be appreciated that any factors upstream, downstream or in
physical association with Factor VIII (e.g. von Willebrand factor) may be
modulated using the present teachings.

[0111] Thus, according to an embodiment of the present invention
modulating transplant organ size comprises enhancing the transplant organ
size. This may be effected using an agent capable of down-regulating an
activity or expression of a coagulation factor or an effector thereof.

[0115] The term "Factor Xa" as used herein refers to coagulation Factor X
or mimetics thereof such as set forth in GenBank Accession Nos.
NM--000504 (SEQ ID NO: 9) and NP--000495 (SEQ ID NO: 10).

[0116] The term "Thrombin" as used herein refers to coagulation Factor IIa
or mimetics thereof such as set forth in GenBank Accession Nos.
NM--000506 (SEQ ID NO: 11) and NP--000497 (SEQ ID NO: 12).

[0117] The phrase "Protease-Activated Receptor (PAR)" as used herein
refers to the seven transmembrane G-protein-coupled receptor, which is
expressed throughout the body (e.g. on platelets, endothelial cells,
myocytes and neurons) and is typically activated by the action of serine
proteases such as thrombin. Examples of PAR receptors include, but are
not limited to, PAR1 e.g. as set forth in GenBank Accession Nos.
NM--001992 and NP--001983, PAR2 e.g. as set forth in GenBank
Accession Nos. NM--005242 and NP--005233, PAR3 e.g. as set
forth in GenBank Accession Nos. NM--004101 and NP--004092 and
PAR4 e.g. as set forth in GenBank Accession Nos. NM--003950 and
NP--003941.

[0120] The phrase "activity or expression of a coagulation factor or an
effector thereof" as used herein refers to the activity of the
coagulation factor or an effector thereof on modulation of transplant
organ size and may be independent of the coagulation activity of the
factor.

[0121] Thus, enhancement in transplant organ size is achieved by
down-regulating the expression level and/or activity of a coagulation
factor or an effector thereof in the subject. Down-regulating the
expression level and/or activity of a coagulation factor or an effector
thereof is preferably effected so as to maximally decrease the expression
level and/or activity of the coagulation factor or an effector thereof in
the subject, so as to achieve optimal enhancement in transplant organ
size. Down-regulating the expression level and/or activity of a
coagulation factor or an effector thereof can be achieved in any of
various ways.

[0122] Downregulation of a coagulation factor or an effector thereof can
be effected on the genomic and/or the transcript level using a variety of
molecules which interfere with transcription and/or translation (e.g.,
RNA silencing agents, Ribozyme, DNAzyme and antisense), or on the protein
level using e.g., antagonists, enzymes that cleave the polypeptide and
the like.

[0123] Following is a list of agents capable of down-regulating expression
level and/or activity of a coagulation factor or an effector thereof.

[0124] Downregulation of a coagulation factor or an effector thereof can
be achieved by RNA silencing. As used herein, the phrase "RNA silencing"
refers to a group of regulatory mechanisms [e.g. RNA interference (RNAi),
transcriptional gene silencing (TGS), post-transcriptional gene silencing
(PTGS), quelling, co-suppression, and translational repression] mediated
by RNA molecules which result in the inhibition or "silencing" of the
expression of a corresponding protein-coding gene. RNA silencing has been
observed in many types of organisms, including plants, animals, and
fungi.

[0125] As used herein, the term "RNA silencing agent" refers to an RNA
which is capable of inhibiting or "silencing" the expression of a target
gene. In certain embodiments, the RNA silencing agent is capable of
preventing complete processing (e.g., the full translation and/or
expression) of an mRNA molecule through a post-transcriptional silencing
mechanism. RNA silencing agents include noncoding RNA molecules, for
example RNA duplexes comprising paired strands, as well as precursor RNAs
from which such small non-coding RNAs can be generated. Exemplary RNA
silencing agents include dsRNAs such as siRNAs, miRNAs and shRNAs. In one
embodiment, the RNA silencing agent is capable of inducing RNA
interference. In another embodiment, the RNA silencing agent is capable
of mediating translational repression.

[0126] RNA interference refers to the process of sequence-specific
post-transcriptional gene silencing in animals mediated by short
interfering RNAs (siRNAs). The corresponding process in plants is
commonly referred to as post-transcriptional gene silencing or RNA
silencing and is also referred to as quelling in fungi. The process of
post-transcriptional gene silencing is thought to be an
evolutionarily-conserved cellular defense mechanism used to prevent the
expression of foreign genes and is commonly shared by diverse flora and
phyla. Such protection from foreign gene expression may have evolved in
response to the production of double-stranded RNAs (dsRNAs) derived from
viral infection or from the random integration of transposon elements
into a host genome via a cellular response that specifically destroys
homologous single-stranded RNA or viral genomic RNA.

[0127] The presence of long dsRNAs in cells stimulates the activity of a
ribonuclease III enzyme referred to as dicer. Dicer is involved in the
processing of the dsRNA into short pieces of dsRNA known as short
interfering RNAs (siRNAs). Short interfering RNAs derived from dicer
activity are typically about 21 to about 23 nucleotides in length and
comprise about 19 base pair duplexes. The RNAi response also features an
endonuclease complex, commonly referred to as an RNA-induced silencing
complex (RISC), which mediates cleavage of single-stranded RNA having
sequence complementary to the antisense strand of the siRNA duplex.
Cleavage of the target RNA takes place in the middle of the region
complementary to the antisense strand of the siRNA duplex.

[0128] Accordingly, the present invention contemplates use of dsRNA to
down-regulate protein expression from mRNA.

[0129] According to one embodiment, the dsRNA is greater than 30 bp. The
use of long dsRNAs (i.e. dsRNA greater than 30 bp) has been very limited
owing to the belief that these longer regions of double stranded RNA will
result in the induction of the interferon and PKR response. However, the
use of long dsRNAs can provide numerous advantages in that the cell can
select the optimal silencing sequence alleviating the need to test
numerous siRNAs; long dsRNAs will allow for silencing libraries to have
less complexity than would be necessary for siRNAs; and, perhaps most
importantly, long dsRNA could prevent viral escape mutations when used as
therapeutics.

[0132] The present invention also contemplates introduction of long dsRNA
specifically designed not to induce the interferon and PKR pathways for
down-regulating gene expression. For example, Shinagwa and Ishii [Genes &
Dev. 17 (11): 1340-1345, 2003] have developed a vector, named pDECAP, to
express long double-strand RNA from an RNA polymerase II (Pol II)
promoter. Because the transcripts from pDECAP lack both the 5'-cap
structure and the 3'-poly(A) tail that facilitate ds-RNA export to the
cytoplasm, long ds-RNA from pDECAP does not induce the interferon
response.

[0133] Another method of evading the interferon and PKR pathways in
mammalian systems is by introduction of small inhibitory RNAs (siRNAs)
either via transfection or endogenous expression.

[0134] The term "siRNA" refers to small inhibitory RNA duplexes (generally
between 18-30 basepairs) that induce the RNA interference (RNAi) pathway.
Typically, siRNAs are chemically synthesized as 21mers with a central 19
bp duplex region and symmetric 2-base 3'-overhangs on the termini,
although it has been recently described that chemically synthesized RNA
duplexes of 25-30 base length can have as much as a 100-fold increase in
potency compared with 21mers at the same location. The observed increased
potency obtained using longer RNAs in triggering RNAi is theorized to
result from providing Dicer with a substrate (27mer) instead of a product
(21mer) and that this improves the rate or efficiency of entry of the
siRNA duplex into RISC.

[0135] It has been found that position of the 3'-overhang influences
potency of a siRNA and asymmetric duplexes having a 3'-overhang on the
antisense strand are generally more potent than those with the
3'-overhang on the sense strand (Rose et al., 2005). This can be
attributed to asymmetrical strand loading into RISC, as the opposite
efficacy patterns are observed when targeting the antisense transcript.

[0136] The strands of a double-stranded interfering RNA (e.g., a siRNA)
may be connected to form a hairpin or stem-loop structure (e.g., a
shRNA). Thus, as mentioned the RNA silencing agent of the present
invention may also be a short hairpin RNA (shRNA).

[0137] The term "shRNA", as used herein, refers to an RNA agent having a
stem-loop structure, comprising a first and second region of
complementary sequence, the degree of complementarity and orientation of
the regions being sufficient such that base pairing occurs between the
regions, the first and second regions being joined by a loop region, the
loop resulting from a lack of base pairing between nucleotides (or
nucleotide analogs) within the loop region. The number of nucleotides in
the loop is a number between and including 3 to 23, or 5 to 15, or 7 to
13, or 4 to 9, or 9 to 11. Some of the nucleotides in the loop can be
involved in base-pair interactions with other nucleotides in the loop.
Examples of oligonucleotide sequences that can be used to form the loop
include 5'-UUCAAGAGA-3' (Brummelkamp, T. R. et al. (2002) Science 296:
550) and 5'-UUUGUGUAG-3' (Castanotto, D. et al. (2002) RNA 8:1454). It
will be recognized by one of skill in the art that the resulting single
chain oligonucleotide forms a stem-loop or hairpin structure comprising a
double-stranded region capable of interacting with the RNAi machinery.

[0138] According to another embodiment the RNA silencing agent may be a
miRNA. miRNAs are small RNAs made from genes encoding primary transcripts
of various sizes. They have been identified in both animals and plants.
The primary transcript (termed the "pri-miRNA") is processed through
various nucleolytic steps to a shorter precursor miRNA, or "pre-miRNA."
The pre-miRNA is present in a folded form so that the final (mature)
miRNA is present in a duplex, the two strands being referred to as the
miRNA (the strand that will eventually basepair with the target). The
pre-miRNA is a substrate for a form of dicer that removes the miRNA
duplex from the precursor, after which, similarly to siRNAs, the duplex
can be taken into the RISC complex. It has been demonstrated that miRNAs
can be transgenically expressed and be effective through expression of a
precursor form, rather than the entire primary form (Parizotto et al.
(2004) Genes & Development 18:2237-2242 and Guo et al. (2005) Plant Cell
17:1376-1386).

[0140] Synthesis of RNA silencing agents suitable for use with the present
invention can be effected as follows. First, the coagulation factor (e.g.
Factor VIII) mRNA sequence is scanned downstream of the AUG start codon
for AA dinucleotide sequences. Occurrence of each AA and the 3' adjacent
19 nucleotides is recorded as potential siRNA target sites. Preferably,
siRNA target sites are selected from the open reading frame, as
untranslated regions (UTRs) are richer in regulatory protein binding
sites. UTR-binding proteins and/or translation initiation complexes may
interfere with binding of the siRNA endonuclease complex [Tuschl
ChemBiochem. 2:239-245]. It will be appreciated though, that siRNAs
directed at untranslated regions may also be effective, as demonstrated
for GAPDH wherein siRNA directed at the 5' UTR mediated about 90%
decrease in cellular GAPDH mRNA and completely abolished protein level
(www.ambion.com/techlib/tn/91/912.html).

[0141] Second, potential target sites are compared to an appropriate
genomic database (e.g., human, mouse, rat etc.) using any sequence
alignment software, such as the BLAST software available from the NCBI
server (www.ncbi.nlm.nih.gov/BLAST/). Putative target sites which exhibit
significant homology to other coding sequences are filtered out.

[0142] Qualifying target sequences are selected as template for siRNA
synthesis. Preferred sequences are those including low G/C content as
these have proven to be more effective in mediating gene silencing as
compared to those with G/C content higher than 55%. Several target sites
are preferably selected along the length of the target gene for
evaluation. For better evaluation of the selected siRNAs, a negative
control is preferably used in conjunction. Negative control siRNA
preferably include the same nucleotide composition as the siRNAs but lack
significant homology to the genome. Thus, a scrambled nucleotide sequence
of the siRNA is preferably used, provided it does not display any
significant homology to any other gene.

[0146] It will be appreciated that the RNA silencing agent of the present
invention need not be limited to those molecules containing only RNA, but
further encompasses chemically-modified nucleotides and non-nucleotides.

[0147] In some embodiments, the RNA silencing agent provided herein can be
functionally associated with a cell-penetrating peptide". As used herein,
a "cell-penetrating peptide" is a peptide that comprises a short (about
12-30 residues) amino acid sequence or functional motif that confers the
energy-independent (i.e., non-endocytotic) translocation properties
associated with transport of the membrane-permeable complex across the
plasma and/or nuclear membranes of a cell. The cell-penetrating peptide
used in the membrane-permeable complex of the present invention
preferably comprises at least one non-functional cysteine residue, which
is either free or derivatized to form a disulfide link with a
double-stranded ribonucleic acid that has been modified for such linkage.
Representative amino acid motifs conferring such properties are listed in
U.S. Pat. No. 6,348,185, the contents of which are expressly incorporated
herein by reference. The cell-penetrating peptides of the present
invention preferably include, but are not limited to, penetratin,
transportan, pIsl, TAT(48-60), pVEC, MTS, and MAP.

[0150] Downregulation of a coagulation factor or an effector thereof can
also be effected by using an antisense polynucleotide capable of
specifically hybridizing with an mRNA transcript encoding a coagulation
factor or an effector thereof (e.g. Factor VIII, Factor X and Thrombin).

[0151] Design of antisense molecules which can be used to efficiently
downregulate a coagulation factor or an effector thereof must be effected
while considering two aspects important to the antisense approach. The
first aspect is delivery of the oligonucleotide into the cytoplasm of the
appropriate cells, while the second aspect is design of an
oligonucleotide which specifically binds the designated mRNA within cells
in a way which inhibits translation thereof.

[0153] In addition, algorithms for identifying those sequences with the
highest predicted binding affinity for their target mRNA based on a
thermodynamic cycle that accounts for the energetics of structural
alterations in both the target mRNA and the oligonucleotide are also
available [see, for example, Walton et al. Biotechnol Bioeng 65: 1-9
(1999)].

[0154] Such algorithms have been successfully used to implement an
antisense approach in cells. For example, the algorithm developed by
Walton et al. enabled scientists to successfully design antisense
oligonucleotides for rabbit beta-globin (RBG) and mouse tumor necrosis
factor-alpha (TNF alpha) transcripts. The same research group has more
recently reported that the antisense activity of rationally selected
oligonucleotides against three model target mRNAs (human lactate
dehydrogenase A and B and rat gp130) in cell culture as evaluated by a
kinetic PCR technique proved effective in almost all cases, including
tests against three different targets in two cell types with
phosphodiester and phosphorothioate oligonucleotide chemistries.

[0155] In addition, several approaches for designing and predicting
efficiency of specific oligonucleotides using an in vitro system were
also published [Matveeva et al., Nature Biotechnology 16: 1374-1375
(1998)].

[0156] For example, a suitable antisense oligonucleotides targeted against
the Factor VIII mRNA (which is coding for the Factor VIII protein) would
be of the following sequences:

[0159] Thus, the current consensus is that recent developments in the
field of antisense technology which, as described above, have led to the
generation of highly accurate antisense design algorithms and a wide
variety of oligonucleotide delivery systems, enable an ordinarily skilled
artisan to design and implement antisense approaches suitable for
down-regulating expression of known sequences without having to resort to
undue trial and error experimentation.

[0160] Another agent capable of down-regulating a coagulation factor or an
effector thereof is a ribozyme molecule capable of specifically cleaving
an mRNA transcript encoding a coagulation factor (e.g. Factor VIII).
Ribozymes are being increasingly used for the sequence-specific
inhibition of gene expression by the cleavage of mRNAs encoding proteins
of interest [Welch et al., Curr Opin Biotechnol. 9:486-96 (1998)]. The
possibility of designing ribozymes to cleave any specific target RNA has
rendered them valuable tools in both basic research and therapeutic
applications. In the therapeutics area, ribozymes have been exploited to
target viral RNAs in infectious diseases, dominant oncogenes in cancers
and specific somatic mutations in genetic disorders [Welch et al., Clin
Diagn Virol. 10:163-71 (1998)]. Most notably, several ribozyme gene
therapy protocols for HIV patients are already in Phase 1 trials. More
recently, ribozymes have been used for transgenic animal research, gene
target validation and pathway elucidation. Several ribozymes are in
various stages of clinical trials. ANGIOZYME was the first chemically
synthesized ribozyme to be studied in human clinical trials. ANGIOZYME
specifically inhibits formation of the VEGF-r (Vascular Endothelial
Growth Factor receptor), a key component in the angiogenesis pathway.
Ribozyme Pharmaceuticals, Inc., as well as other firms have demonstrated
the importance of anti-angiogenesis therapeutics in animal models.
HEPTAZYME, a ribozyme designed to selectively destroy Hepatitis C Virus
(HCV) RNA, was found effective in decreasing Hepatitis C viral RNA in
cell culture assays (Ribozyme Pharmaceuticals, Incorporated--WEB home
page).

[0161] An additional method of regulating the expression of Factor VIII
gene and/or genes of other coagulation factors in cells is via triplex
forming oligonucleotides (TFOs). Recent studies have shown that TFOs can
be designed which can recognize and bind to polypurine/polypirimidine
regions in double-stranded helical DNA in a sequence-specific manner.
These recognition rules are outlined by Maher III, L. J., et al.,
Science, 1989; 245:725-730; Moser, H. E., et al., Science, 1987;
238:645-630; Beal, P. A., et al, Science, 1992; 251:1360-1363; Cooney,
M., et al., Science, 1988; 241:456-459; and Hogan, M. E., et al., EP
Publication 375408. Modification of the oligonucleotides, such as the
introduction of intercalators and backbone substitutions, and
optimization of binding conditions (pH and cation concentration) have
aided in overcoming inherent obstacles to TFO activity such as charge
repulsion and instability, and it was recently shown that synthetic
oligonucleotides can be targeted to specific sequences (for a recent
review see Seidman and Glazer, J Clin Invest 2003; 112:487-94).

[0162] In general, the triplex-forming oligonucleotide has the sequence
correspondence:

[0163] However, it has been shown that the A-AT and G-GC triplets have the
greatest triple helical stability (Reither and Jeltsch, BMC Biochem,
2002, Sep. 12, Epub). The same authors have demonstrated that TFOs
designed according to the A-AT and G-GC rule do not form non-specific
triplexes, indicating that the triplex formation is indeed sequence
specific.

[0164] Thus for any given sequence in the Factor VIII regulatory region
(or the regulatory region of other coagulation factors or effectors
thereof) a triplex forming sequence may be devised. Triplex-forming
oligonucleotides preferably are at least 15, more preferably 25, still
more preferably 30 or more nucleotides in length, up to 50 or 100 bp.

[0166] Additionally, TFOs designed according to the abovementioned
principles can induce directed mutagenesis capable of effecting DNA
repair, thus providing both downregulation and upregulation of expression
of endogenous genes (Seidman and Glazer, J Clin Invest 2003; 112:487-94).
Detailed description of the design, synthesis and administration of
effective TFOs can be found in U.S. Patent Application Nos. 2003 017068
and 2003 0096980 to Froehler et al, and 2002 0128218 and 2002 0123476 to
Emanuele et al, and U.S. Pat. No. 5,721,138 to Lawn.

[0167] Downregulation of a coagulation factor or an effector thereof can
also be effected at the protein level using e.g., antagonists, enzymes.
For example, Factor VIII can be down-regulated by, for example, Factor
VIII antagonists [e.g. TB-402 (Thromb-X NV)] or Factor VIII inhibitory
peptide (e.g. Factor VIII neutralizing antibody). Downregulation of
Factor X can be effected using, for example, Clexane, JTV-803 or
Fondaparinux. Downregulation of Thrombin can be effected using, for
example, Clexane, Dabigatran, Hirudin, Bivalirudin, Lepirudin, Desirudin,
Argatroban, Melagatran or ximelagatran.

[0168] Another agent which can be used along with the present invention to
down-regulate a coagulation factor or an effector thereof is a molecule
which prevents a coagulation factor's (e.g. Factor VIII) activation or
substrate binding. Such a molecule may comprise an antibody which
specifically binds Factor VIII, as for example, sc-73597 [Santa Cruz
Biotechnology] or F4.55, F4.77, F4.264, F4.115 and F4.415 [Sola et al.,
PNAS (1982) 79 (1) 183-187] Likewise, antibodies which specifically
target Factor X (e.g. ab61361, Abcam) or Thrombin (e.g. sc-59716,
sc-80590, sc-73475, sc-59717, sc-59718, sc-65961, Santa Cruz
Biotechnology) may be used according to the present teachings.

[0169] According to a specific embodiment of the present invention,
synthetic peptides or antibodies which inhibit PARs (directed at e.g.
PAR1, PAR2, PAR3 or PAR4) may also be used to downregulate PAR signaling,
such as for example, the PAR1 agonist TFLLR-NH2 (SEQ ID NO: 13), the PAR4
agonist AYPGKF-NH2 (SEQ ID NO: 14), the palmitoylated peptides
pal-RCLSSSAVANRS (SEQ ID NO: 15, PAR1 antagonist) and pal-SGRRYGHALR (SEQ
ID NO: 16, PAR4 antagonist). Such peptide antagonists may be generated by
any method known to one of ordinary skill in the art, such as by
solid-phase peptide synthesis using in situ neutralization/HBTU by Hadar
Biotec, Israel.

[0170] It will be appreciated that downregulation of a coagulation factor
or an effector thereof can also be effected by up-regulating the activity
or expression of antithrombin or Protein C.

[0171] It will be appreciated that according to the present teachings
Vitamin K levels may also be modulated to enhance or decrease organ size.

[0172] It will be appreciated that in order to increase organ size (e.g.
spleen), G-CSF may be administered prior to, concomitantly with, or
following administration of the above described agents (e.g. Clexane)
Likewise, other growth factor and/or cytokines may be administered to the
subject to modulate organ size including, but not limited to, Hepatocyte
growth factor (HGF) and Keratinocyte growth factor (KGF).

[0173] In some instances decreasing organ size may be desirable while
maintaining functionality. For instance, kidney transplantation from an
adult to an infant may be desired or decreasing splenomegaly of a
transplanted organ. It will be appreciated that according to the present
teachings, decreasing transplant organ size is achieved by up-regulating
the expression level and/or activity of a coagulation factor or an
effector thereof in the subject. Up-regulating the expression level
and/or activity of a coagulation factor or an effector thereof is
preferably effected so as to maximally increase the expression level
and/or activity of a coagulation factor or an effector thereof in the
subject, so as to achieve optimal decrease in transplant organ size.
Up-regulating the expression level and/or activity of a coagulation
factor or an effector thereof can be achieved in any of various ways.

[0175] It will be appreciated that decreasing an organ size can also be
effected by down-regulating the activity or expression of anti-thrombin.

[0176] According to an exemplary embodiment of the present invention,
modulating the expression level and/or activity of a coagulation factor
or an effector thereof may be effected prior to, concomitantly with or
following transplantation of an organ. Thus, modulating the expression
level and/or activity of a coagulation factor or an effector thereof is
effected so as to maximally enable organ engraftment into the subject
with minimal organ failure.

[0177] Each of the agents used for up-regulating or down-regulating
coagulation factor or an effector thereof described hereinabove can be
administered to the subject per se or as part of a pharmaceutical
composition which also includes a physiologically acceptable carrier. The
purpose of a pharmaceutical composition is to facilitate administration
of the active ingredient to an organism.

[0178] It will be appreciated that the pharmaceutical composition may
further comprise an immunosuppressive agent as described in detail
hereinabove.

[0179] As used herein a "pharmaceutical composition" refers to a
preparation of one or more of the active ingredients described herein
with other chemical components such as physiologically suitable carriers
and excipients. The purpose of a pharmaceutical composition is to
facilitate administration of a compound to an organism.

[0180] Herein the term "active ingredient" refers to the coagulation
factor or an effector thereof thereof upregulating or downregulating
agents accountable for the biological effect.

[0181] Hereinafter, the phrases "physiologically acceptable carrier" and
"pharmaceutically acceptable carrier" which may be interchangeably used
refer to a carrier or a diluent that does not cause significant
irritation to an organism and does not abrogate the biological activity
and properties of the administered compound. An adjuvant is included
under these phrases.

[0182] Herein the term "excipient" refers to an inert substance added to a
pharmaceutical composition to further facilitate administration of an
active ingredient. Examples, without limitation, of excipients include
calcium carbonate, calcium phosphate, various sugars and types of starch,
cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0183] Techniques for formulation and administration of drugs may be found
in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton,
Pa., latest edition, which is incorporated herein by reference.

[0184] Suitable routes of administration may, for example, include oral or
parenteral delivery, including intramuscular, subcutaneous and
intramedullary injections as well as intrathecal, direct
intraventricular, intracardiac, e.g., into the right or left ventricular
cavity, into the common coronary artery, intravenous, inrtaperitoneal,
intranasal, or intraocular injections.

[0185] Alternately, one may administer the pharmaceutical composition in a
local rather than systemic manner, for example, via injection of the
pharmaceutical composition directly into a tissue region of a patient.

[0187] Pharmaceutical compositions of the present invention may be
manufactured by processes well known in the art, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making, levigating,
emulsifying, encapsulating, entrapping or lyophilizing processes.

[0188] Pharmaceutical compositions for use in accordance with the present
invention thus may be formulated in conventional manner using one or more
physiologically acceptable carriers comprising excipients and
auxiliaries, which facilitate processing of the active ingredients into
preparations which, can be used pharmaceutically. Proper formulation is
dependent upon the route of administration chosen.

[0189] For injection, the active ingredients of the pharmaceutical
composition may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hank's solution, Ringer's
solution, or physiological salt buffer. For transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art.

[0190] For oral administration, the pharmaceutical composition can be
formulated readily by combining the active compounds with
pharmaceutically acceptable carriers well known in the art. Such carriers
enable the pharmaceutical composition to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the
like, for oral ingestion by a patient. Pharmacological preparations for
oral use can be made using a solid excipient, optionally grinding the
resulting mixture, and processing the mixture of granules, after adding
suitable auxiliaries if desired, to obtain tablets or dragee cores.
Suitable excipients are, in particular, fillers such as sugars, including
lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as,
for example, maize starch, wheat starch, rice starch, potato starch,
gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose,
sodium carbomethylcellulose; and/or physiologically acceptable polymers
such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may
be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic
acid or a salt thereof such as sodium alginate.

[0191] Dragee cores are provided with suitable coatings. For this purpose,
concentrated sugar solutions may be used which may optionally contain gum
arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol,
titanium dioxide, lacquer solutions and suitable organic solvents or
solvent mixtures. Dyestuffs or pigments may be added to the tablets or
dragee coatings for identification or to characterize different
combinations of active compound doses.

[0192] Pharmaceutical compositions which can be used orally, include
push-fit capsules made of gelatin as well as soft, sealed capsules made
of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit
capsules may contain the active ingredients in admixture with filler such
as lactose, binders such as starches, lubricants such as talc or
magnesium stearate and, optionally, stabilizers. In soft capsules, the
active ingredients may be dissolved or suspended in suitable liquids,
such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In
addition, stabilizers may be added. All formulations for oral
administration should be in dosages suitable for the chosen route of
administration.

[0193] For buccal administration, the compositions may take the form of
tablets or lozenges formulated in conventional manner.

[0194] For administration by nasal inhalation, the active ingredients for
use according to the present invention are conveniently delivered in the
form of an aerosol spray presentation from a pressurized pack or a
nebulizer with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichloro-tetrafluoroethane or carbon dioxide. In the case of a
pressurized aerosol, the dosage unit may be determined by providing a
valve to deliver a metered amount. Capsules and cartridges of, e.g.,
gelatin for use in a dispenser may be formulated containing a powder mix
of the compound and a suitable powder base such as lactose or starch.

[0195] The pharmaceutical composition described herein may be formulated
for parenteral administration, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit dosage
form, e.g., in ampoules or in multidose containers with optionally, an
added preservative. The compositions may be suspensions, solutions or
emulsions in oily or aqueous vehicles, and may contain formulatory agents
such as suspending, stabilizing and/or dispersing agents.

[0196] Pharmaceutical compositions for parenteral administration include
aqueous solutions of the active preparation in water-soluble form.
Additionally, suspensions of the active ingredients may be prepared as
appropriate oily or water based injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame oil, or
synthetic fatty acids esters such as ethyl oleate, triglycerides or
liposomes. Aqueous injection suspensions may contain substances, which
increase the viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol or dextran. Optionally, the suspension may also
contain suitable stabilizers or agents which increase the solubility of
the active ingredients to allow for the preparation of highly
concentrated solutions.

[0197] Alternatively, the active ingredient may be in powder form for
constitution with a suitable vehicle, e.g., sterile, pyrogen-free water
based solution, before use.

[0198] The pharmaceutical composition of the present invention may also be
formulated in rectal compositions such as suppositories or retention
enemas, using, e.g., conventional suppository bases such as cocoa butter
or other glycerides.

[0199] Pharmaceutical compositions suitable for use in context of the
present invention include compositions wherein the active ingredients are
contained in an amount effective to achieve the intended purpose. More
specifically, a therapeutically effective amount means an amount of
active ingredients (coagulation factor or an effector thereof
upregulating or downregulating agents) effective to modulate transplant
organ size of the subject being treated.

[0200] Determination of a therapeutically effective amount is well within
the capability of those skilled in the art, especially in light of the
detailed disclosure provided herein.

[0201] For any preparation used in the methods of the invention, the
therapeutically effective amount or dose can be estimated initially from
in vitro and cell culture assays. For example, a dose can be formulated
in animal models to achieve a desired concentration or titer. Such
information can be used to more accurately determine useful doses in
humans.

[0202] Toxicity and therapeutic efficacy of the active ingredients
described herein can be determined by standard pharmaceutical procedures
in vitro, in cell cultures or experimental animals. The data obtained
from these in vitro and cell culture assays and animal studies can be
used in formulating a range of dosage for use in human. The dosage may
vary depending upon the dosage form employed and the route of
administration utilized. The exact formulation, route of administration
and dosage can be chosen by the individual physician in view of the
patient's condition. (See e.g., Fingl, et al., 1975, in "The
Pharmacological Basis of Therapeutics", Ch. 1 p. 1).

[0203] Dosage amount and interval may be adjusted individually to provide
adequate levels of the active ingredient as to induce or suppress the
biological effect (minimal effective concentration, MEC). The MEC will
vary for each preparation, but can be estimated from in vitro data.
Dosages necessary to achieve the MEC will depend on individual
characteristics and route of administration. Detection assays can be used
to determine plasma concentrations.

[0204] Depending on the severity and responsiveness of the condition to be
treated, dosing can be of a single or a plurality of administrations,
with course of treatment lasting from several days to several weeks or
until cure is effected or diminution of the disease state is achieved.

[0205] The amount of a composition to be administered will, of course, be
dependent on the subject being treated, the severity of the affliction,
the manner of administration, the judgment of the prescribing physician,
etc.

[0206] The modulating factors will be given for a sufficient amount of
time to enable modulation of organ transplant size without compromising
blood coagulation levels (e.g. bleeding or blood clot formation) in the
subject. Thus, it is advisable to draw a base-line blood sample from each
subject prior to administration of the modulating agents of the present
invention. Furthermore, once a subject received modulating factors, it is
advisable that they return for follow-up evaluation, which include, for
example, hematologic and chemical tests for safety.

[0207] Compositions of the present invention may, if desired, be presented
in a pack or dispenser device, such as an FDA approved kit, which may
contain one or more unit dosage forms containing the active ingredient.
The pack may, for example, comprise metal or plastic foil, such as a
blister pack. The pack or dispenser device may be accompanied by
instructions for administration. The pack or dispenser may also be
accommodated by a notice associated with the container in a form
prescribed by a governmental agency regulating the manufacture, use or
sale of pharmaceuticals, which notice is reflective of approval by the
agency of the form of the compositions or human or veterinary
administration. Such notice, for example, may be of labeling approved by
the U.S. Food and Drug Administration for prescription drugs or of an
approved product insert. Compositions comprising a preparation of the
invention formulated in a compatible pharmaceutical carrier may also be
prepared, placed in an appropriate container, and labeled for treatment
of an indicated condition, as is further detailed above.

[0209] It is expected that during the life of a patent maturing from this
application many relevant upregulating or downregulating agents for
coagulation factors or effectors thereof will be developed and the scope
of the term coagulation factor or an effector thereof upregulating or
downregulating agents is intended to include all such new technologies a
priori.

[0213] The term "consisting essentially of" means that the composition,
method or structure may include additional ingredients, steps and/or
parts, but only if the additional ingredients, steps and/or parts do not
materially alter the basic and novel characteristics of the claimed
composition, method or structure.

[0214] As used herein, the singular form "a", "an" and "the" include
plural references unless the context clearly dictates otherwise. For
example, the term "a compound" or "at least one compound" may include a
plurality of compounds, including mixtures thereof.

[0215] Throughout this application, various embodiments of this invention
may be presented in a range format. It should be understood that the
description in range format is merely for convenience and brevity and
should not be construed as an inflexible limitation on the scope of the
invention. Accordingly, the description of a range should be considered
to have specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example, description
of a range such as from 1 to 6 should be considered to have specifically
disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2
to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within
that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of
the breadth of the range.

[0216] Whenever a numerical range is indicated herein, it is meant to
include any cited numeral (fractional or integral) within the indicated
range. The phrases "ranging/ranges between" a first indicate number and a
second indicate number and "ranging/ranges from" a first indicate number
"to" a second indicate number are used herein interchangeably and are
meant to include the first and second indicated numbers and all the
fractional and integral numerals therebetween.

[0217] As used herein the term "method" refers to manners, means,
techniques and procedures for accomplishing a given task including, but
not limited to, those manners, means, techniques and procedures either
known to, or readily developed from known manners, means, techniques and
procedures by practitioners of the chemical, pharmacological, biological,
biochemical and medical arts.

[0218] As used herein, the term "treating" includes abrogating,
substantially inhibiting, slowing or reversing the progression of a
condition, substantially ameliorating clinical or aesthetical symptoms of
a condition or substantially preventing the appearance of clinical or
aesthetical symptoms of a condition.

[0219] It is appreciated that certain features of the invention, which
are, for clarity, described in the context of separate embodiments, may
also be provided in combination in a single embodiment. Conversely,
various features of the invention, which are, for brevity, described in
the context of a single embodiment, may also be provided separately or in
any suitable subcombination or as suitable in any other described
embodiment of the invention. Certain features described in the context of
various embodiments are not to be considered essential features of those
embodiments, unless the embodiment is inoperative without those elements.

[0220] Various embodiments and aspects of the present invention as
delineated hereinabove and as claimed in the claims section below find
experimental support in the following examples.

EXAMPLES

[0221] Reference is now made to the following examples, which together
with the above descriptions, illustrate the invention in a non limiting
fashion.

Involvement of Factor VIII in the Control of Transplant Organ Size During
Transplantation of Embryonic Porcine Grafts into SCID Mice

[0223] Materials and Experimental Procedures

[0224] Animals

[0225] All animals were maintained under conditions approved by the
Institutional Animal Care and Use Committee at the Weizmann Institute.
The study protocol was approved by the ethics committees at Kibbutz Lahav
and the Weizmann Institute.

[0227] In addition, immune deficient RAG.sup.-/- mice or RAG.sup.-/- FVIII
KO mice were used as hosts for the transplantation studies. To obtain
RAG.sup.-/- hemophilic mice (designated RAG.sup.-/- FVIII KO mice), FVIII
mutation was introduced into RAG.sup.-/- mice, as was previously
described. All mice were kept in small cages (up to five animals per
cage) and fed sterile food.

[0228] Pig embryos were obtained from the Lahav Institute of Animal
Research (Kibbutz Lahav, Israel). Pregnant sows were operated on at
embryonic days 28 (E28), for liver tissue, and 42 (E42), for spleen and
pancreatic tissues, under general anesthesia. Warm ischemia time was less
than 10 minutes and the embryos were transferred to cold PBS. Spleen,
pancreas and liver precursors for transplantation were extracted under a
light microscope and were kept in sterile conditions at 4° C. in
RPMI 1640 (Biological Industries, Beit HaEmek, Israel) prior to
transplantation. Cold ischemia time until transplantation was less than 2
hours.

[0229] Mouse embryos were obtained from C57BL/6 pregnant female mice.
Pregnant mice were operated on at embryonic days 15 (E15), for spleen
tissue, and 16 (E16), for liver and pancreatic tissues, under general
anesthesia. Warm ischemia time was less than 10 minutes and the embryos
were transferred to cold PBS. Spleen, pancreas and liver precursors for
transplantation were extracted under a light microscope and were kept in
sterile conditions at 4° C. in RPMI 1640 (Biological Industries,
Beit HaEmek, Israel) prior to transplantation. Cold ischemia time until
transplantation was less than 2 hours.

[0230] Transplantation Procedure

[0231] Transplantation of pig precursors was completed as previously
described [Dekel, B. et al., Nat Med (2003) 9, 53-60]. Briefly,
transplantation of the embryonic precursors was performed under general
anesthesia (2.5% 2,2,2-Tribromoethanol, 97% in PBS, 10 ml/kg
intraperitoneally). Host kidney was exposed through a left lateral
incision. A 1.5-mm incision was made at the caudal end of the kidney
capsule and donor precursors were grafted under the kidney capsule in
fragments 1-2 mm in diameter.

[0232] Morphometric Analysis

[0233] Three months post transplantation E42 pancreatic grafts were
formalin fixed and embedded in paraffin. Consecutive 40 μm sections
were cut and stained. The areas of interest were quantified using the
Image Pro program (Media Cybernetics).

[0234] ELISA Measurements of Pig Insulin and Albumin

[0235] A porcine/human insulin kit (Catalog No. K6219, DAKO), in which the
primary pig anti-insulin antibody does not cross-react with mouse
insulin, was used to follow pig insulin levels according to the
manufacturer's instructions.

[0239] Paraffin sections (4μ) were xylene deparaffinized and
rehydrated. Endogenous peroxidase was blocked with 0.3% H2O2 in 70%
methanol for 10 minutes. Antigen-retrieval procedures were performed
according to the glucagon antibody manufacturer's instructions. After
blocking, both paraffin sections and 6-μ cryosections were incubated
with specific first antibody for 60 minutes. Detection of antibody
binding was performed by using the following secondary reagents: DAKO
peroxidase EnVision system for the detection of mouse and rabbit
antibodies and Sigma biotinylated anti-goat antibody (followed by extra
avidin peroxidase reagent) for goat antibodies. In all cases,
diaminobenzidine was used as a chromogen.

[0242] Comparisons between groups were evaluated by the Student's t-test.
Data were expressed as mean±SD and were considered statistically
significant at p values of 0.05 or less.

[0243] Results

[0244] Porcine Spleen

[0245] The inventors of the present invention have unexpectedly discovered
that transplanted pig embryonic tissues grew to a larger size in
immunodeficient hemophilic (Factor VIII KO-SCID) recipient mice in
comparison non-hemophilic NOD-SCID mice. As illustrated in FIGS. 1A-B,
three months post transplant of pig embryonic spleen tissue in a Factor
VIII KO SCID recipient mouse, the implanted spleen displayed a typical
oversize versus the size of the implant grown in a factor VIII positive
SCID recipient. The total average weight of the implants was 6.78±2.16
gr for Factor VIII KO-SCID mice compared to 1.46±0.82 gr for SCID mice
(FIG. 1C), suggesting enhancement of spleen size by a factor of 4
(p<0.05). Histological examination of the growing spleen implants
revealed normal growth, development and vascularization patterns
comparable to those found in the corresponding factor VIII wild type mice
(FIGS. 1D-G), ruling out the potential induction of a malignant process.
Together this data indicated a potential involvement of mouse factor VIII
in size control of pig embryonic spleen implants.

[0246] Porcine Liver and Pancreas

[0247] To generalize this finding, inventors further evaluated the growth
of pig embryonic liver and pancreas precursor tissues harvested at a
previously defined optimal `window`, namely E28 and E42, respectively
[Eventov-Friedman, S. et al., Proc Natl Acad Sci USA (2005) 102,
2928-33]. The growth of pancreatic tissues was determined in the SCID
recipient mice by monitoring blood levels of pig insulin using a specific
ELISA previously shown to correlate with implant size [Eventov-Friedman,
S. et al., supra]. As can be seen in FIG. 2A, pig insulin blood levels
were markedly enhanced in Factor VIII KO SCID mice compared to Factor
VIII positive SCID mice, reflecting a growth advantage in the Factor VIII
KO recipients. However, these differences in insulin levels could be
attributed to differences in functionality of β-cells rather than to
the total number of β-cells. Therefore, the total volume of the
implants as well as the fraction of β-insulin positive cells (out of
the total volume) were examined using morphometric analysis (FIG. 2B). As
is illustrated in FIG. 2B, enhancement of pig pancreas size was found to
closely correspond to the difference in insulin blood levels. Thus, the
total volume of insulin positive cells was 0.75±0.003 mm3 in
factor VIII SCID versus 0.23±0.003 mm3 in non-hemophilic SCID
recipients, respectively, suggesting an overall enhancement of implant
size at least by a factor of three (p<0.05).

[0248] Similarly to previously described results [Eventov-Friedman, S. et
al., PLoS Med (2006) 3, e215], E42 pancreatic tissue implanted into
NOD-SCID mice were shown to predominantly comprise endocrine tissue with
minimal exocrine activity. Only a minimal number of exocrine cells were
detected in the E42 graft three months after transplantation, while most
of the cells were of the endocrine lineage. Moreover, the endocrine
compartment architecture of the growing pig pancreas was similar for both
wild type (FIG. 2C) and Factor VIII KO recipient mice (FIG. 2D).

[0249] A significant enhancement in implant size (by at least a factor of
two) was also established upon implantation of embryonic pig liver into
factor VIII KO SCID mice, as evaluated by ELISA for pig albumin blood
levels (FIG. 3A). Again, despite the enhanced growth, the growing pig
liver exhibited similar architecture in both types of recipients (FIGS.
3B-G).

[0250] Taken together, these results strongly suggest that mouse factor
VIII plays a critical role in controlling the size of embryonic pig
implants.

[0252] It is nevertheless possible that the enhancement of organ size
following implantation of pig embryonic spleen, pancreas and liver, might
be related to early events associated with graft accommodation and
vasculature formation, which might potentially differ in Factor VIII KO
versus non-hemophilic SCID recipients and could be independent of the
origin of the donor tissue. To address this possibility, similar
implantation experiments were repeated using embryonic tissues from
C57BL/6 mouse donors. In contrast to the pig implants, no differences in
organ size were found between SCID factor VIII KO and non-hemophilic SCID
recipients following implantation of mouse E15-16 gestational age tissues
(data not shown).

[0253] Spleen grafts of mouse origin exhibited an average size of
1.5±0.35 mm3 three months post transplantation in both SCID and
SCID Factor VIII KO recipients. Similar results were obtained for mouse
embryonic pancreas and liver transplants (data not shown). Furthermore,
no differences in size were found following transplantation of embryonic
mouse tissues obtained from hemophilic donors into factor VIII KO-SCID
versus non-hemophilic SCID mice. Thus, these results suggest that while
the final size of heterologous embryonic pig implants is affected by the
presence or absence of mouse Factor VIII, embryonic mouse implants attain
their final size regardless of the presence of mouse factor VIII.

[0255] Prkdcscid (commonly referred to as SCID) is a spontaneously
occurring mutation in chromosome 16. Furthermore, the Prkdcscid
mutation was backcrossed onto the NOD/ShiLt background to obtain the
NOD-SCID mice. NOD-SCID mice are characterized by an absence of
functional T cells and B cells, lymphopenia, hypogammaglobulinemia and a
normal hematopoietic microenvironment. On the other hand, hemophilic
(Factor VIII KO) mice are homozygous for the targeted, X
chromosome-linked mutant allele, by a neo cassette which was used to
disrupt exon 16 of Factor VIII gene. As previously described [Aronovich
A. et al., Proc Natl Acad Sci USA (2006) 103: 19075-80], by backcrossing
these two strains a new strain was developed of Factor VIII KO mice on a
background of NOD-SCID mice.

[0256] Despite the fact that the SCID mutation in NOD mice is on
chromosome 16 and hemophilia is a chromosome X linked mutation, a risk of
chromosome translocation exists, as in the case of Philadelphia
chromosome abnormality that is associated with chronic myelogenous
leukemia. Therefore, in order to rule out potential artifacts due to
genetic abnormalities in NOD-SCID FVIII KO mice, inventors have attempted
to introduce the Factor VIII KO mutation into a different SCID mouse,
namely, SCID with a RAG.sup.-/- background which was previously shown to
exhibit a targeted mutation on chromosome 2, associated with a
"non-leaky" severe combined immune deficiency. Thus, a new RAG.sup.-/-
FVIII KO colony was established.

[0257] As can be seen in FIGS. 3H-I, RAG.sup.-/- FVIII KO recipients of an
E42 pig spleen implant exhibit at twelve weeks post transplantation an
oversized spleen implant compared to their non-hemophilc RAG.sup.-/-
counterparts.

[0261] 8 to 10 week old immune competent C57BL mice and C57BL hemophilic
(C57BL Hem F8) mice were used for the G-CSF studies.

[0262] All mice were kept in small cages (up to five animals per cage) and
fed sterile food.

[0263] G-CSF Treatment

[0264] 8-10 weeks old NOD-SCID or Factor VIII KO SCID mice were treated by
daily subcutaneous injections of recombinant human G-CSF (Neupogen,
Amgen) at a dose of 250 μg per kg per day for 7 days. To determine the
spleen weight, 7 days after the initiation of G-CSF treatment, mice were
euthanized and spleens were harvested.

[0271] As depicted in detail above, Factor VIII deficiency had no effect
on organ size of mouse embryonic transplants, thus suggesting that the
role of Factor VIII may be limited to a checkpoint of excessive growth
which only operates upon implantation of tissues from a larger organism,
such as a pig. More specifically, it is possible that Factor VIII is
involved in interference in expression and/or activity of a putative
survival factor and thereby acts to define the maximum tolerable tissue
mass. Thus, assuming that mouse and pig embryonic implants are endowed
with stem cell pools of different sizes prior to transplantation, they
are likely to exhibit different organ size upon completion of growth and
differentiation in the mouse recipients (FIGS. 4A-B). Consequently, in
hemophilic mice (e.g. SCID Factor VIII KO mice) lacking the potential
inhibitory activity (i.e. overgrowth checkpoint) mediated by Factor VIII,
the size of the pig transplanted organ size is larger, while mouse
implants growing to the expected mouse size do not exhibit excessive
growth and therefore are not subject to Factor VIII control (FIG. 4C).

[0272] It could be argued that the role of Factor VIII might be limited to
the transplantation setting and therefore might not have a true
physiological role in the intact animal. To test the potential control by
Factor VIII on oversized organs in a more physiological setting,
inventors have further tested the role of Factor VIII in controlling
G-CSF mediated splenomegaly, known to occur within 7 days of infusion of
this agent [Takamatsu, Y. et al., Transfusion (2007) 47, 41-9;
Platzbecker, U. et al., Transfusion (2001) 41, 184-9]. As is illustrated
in FIGS. 5A-B, treatment of Factor VIII KO SCID mice with G-CSF was
associated with a significant enhancement in spleen size (i.e.
splenomegaly) compared to non-hemophilic mice. Thus, the average spleen
weight in the former group was 2.6 fold larger (314.89±121.51 mg,
compared to 118.5±25.56 mg in the latter group, P<0.0001).

[0273] The capacity of exogenous Factor VIII infusion to inhibit the
enhanced splenomegaly in SCID FVIII KO mice under G-CSF stimulation, is
shown in FIG. 5B. Considering that the half life of Hu Factor VIII in the
blood of infused mice was only about four hours, a continuous
administration of Factor VIII (by osmotic pumps) was used in these mice.
Although only about 10% of physiologically circulating Factor VIII was
detected in the plasma of the infused mice, these SCID FVIII KO
recipients exhibited reduced splenomegaly under G-CSF stimulation,
compared to mice not receiving Factor VIII (FIG. 5B, P=0.033).

[0275] While investigation of implant size upon transplantation of pig
embryonic tissue required the use of Factor VIII KO hemophilic SCID mice,
the SCID background is not necessary for the evaluation of splenomegaly
in G-CSF treated mice. Indeed, examination of the splenomegaly revealed a
role for Factor VIII in oversized control also in fully immune competent
Factor VIII KO C57BL/6 hemophilic mice (FIG. 6). Examination of the
enlarged spleens revealed normal growth and development comparable to
that found in the Factor VIII wild type counterpart mice, without any
indication of malignancy.

[0276] As can be seen in FIG. 6, treatment with G-CSF of C57BL hemophilic
(Hem) F8 mice was associated with significantly enhanced splenomegaly,
compared to that found in the non-hemophilic counterparts (average spleen
weight in the former group was 256.03±149.86 mg, compared to
181.22±61.43 mg in the latter group, P=0.0165), upon infusion of the
same doses of G-CSF.

[0277] Taken together these results suggest that Factor VIII is involved
in regulation of organ size in situations by which there is a drive for
oversized growth, either due to large stem cell pool as in the case of
embryonic organ transplantation (from a large transplant donor) or upon
infusion of a cytokine such as G-CSF.

[0285] All experiments were performed using Affymetrix porcine genome
oligonucleotide arrays, as described at:
http://www(dot)affymetrix(dot)com/support/technical/datasheets/porcine_da-
tasheet(dot)pdf.

[0286] Total RNA from each sample was used to prepare biotinylated target
RNA, using minor modifications as recommended by the manufacturer at:
http://www(dot)affymetrix(dot)com/support/technical/manual/expression_man-
ual(dot)affx.

[0287] Briefly, 5 μg of mRNA was used to generate first-strand cDNA by
using a T7-linked oligo(dT) primer. After second-strand synthesis, in
vitro transcription was performed with biotinylated UTP and CTP
(Affymetrix), resulting in approximately 300-fold amplification of RNA.

[0288] The target cDNA generated from each sample was processed as per
manufacturer's recommendation using an Affymetrix GeneChip Instrument
System: http://www(dot)affymetrix(dot)com/support/technical/manual/expres-
sion_manual(dot)affx.

[0289] Briefly, spike controls were added to 15 μg fragmented cRNA
before overnight hybridization. Arrays were then washed and stained with
streptavidin-phycoerythrin, before being scanned on an Affymetrix
GeneChip scanner. A complete description of these procedures is available
at: http://www(dot)affymetrix(dot)com/support/technical/manual/expression-
_manual(dot)affx.

[0290] Additionally, quality and amount of starting RNA was confirmed
using an agarose gel. After scanning, array images were assessed by eye
to confirm scanner alignment and the absence of significant bubbles or
scratches on the chip surface. 3'/5' ratios for GAPDH and beta-actin were
confirmed to be within acceptable limits (3.16-3.4 and 0.38-0.4), and
BioB spike controls were found to be present on all chips, with BioC,
BioD and CreX also present in increasing intensity. When scaled to a
target intensity of 150 (using Affymetrix MAS 5.0 array analysis
software), scaling factors for all arrays were within acceptable limits
(1.62-1.69), as were background, Q values and mean intensities. Details
of quality control measures can be found at:

[0297] The probe sets contained in the Affymetrix porcine genome
oligonucleotide array signals were calculated using Mas 5 algorithm. Pig
implants affected by the difference in mouse Factor VIII expression (in
the different SCID recipients) were compared. The comparison generated a
list of "active genes" representing probe sets changed by at least 2 fold
as calculated from the MAS 5 Log Ratio values(LR>=1 or LR<=-1) and
detected as "Increased" or as "Decrease"(I or D, p-value 0.0025) or
"Marginal Increased" or as or "Marginal Decrease" (MI or MD, p-value
0.003) in all treated sample as compared to all the control samples in at
least one time point. This list excluded up-regulated genes in all
treated samples with signals lower than 20 or detected as absent, and
down regulated gene with base line signals lower than 20 and detected as
absent in the control samples.

[0298] For further filtering we used the probe sets changed by at least 2
fold (between signals) between the x treated samples at x.

[0299] Hierarchical clustering was performed using Spotfire DecisionSite
for Functional Genomics (Somerville, Mass.).

[0305] Functional classifications with an "Ease score" lower than 0.05
were marked as over represented.

[0306] Statistical Analysis

[0307] As described in detail in Example 1, hereinabove.

[0308] Results

[0309] To gain insight into the potential intrinsic genes affected in the
pig embryonic implants transplanted into hemophilic and non-hemophilic
mice, inventors examined global gene expression using DNA microarray
analysis. DNA microarray analysis was carried out for pig spleen implants
in Factor VIII wild type and KO host mice at 8 weeks post transplant
(data not shown). Differential expression of genes which are generally
involved in cell growth, proliferation and apoptosis was demonstrated.
However, differential expression of two gene families, the first
associated with coagulation and endothelium and the second associated
with patterning and differentiation, may be specifically relevant to the
regulation of transplant organ size. For the complete data and discussion
of the most potentially relevant genes identified by this analysis see
Table 2, below.

[0310] Several intrinsic pathways in the pig implants were affected by the
absence of mouse Factor VIII (see Tables 1 and 2). For example, Wnt
pathway components, such as Wnt5b, LRP5 and LRP6, were expressed at
higher levels in spleens grown in Factor VIII KO SCID mice. The Wnt
pathway was previously suggested to be an important regulator of organ
size, for example Suksaweang et al. demonstrated that overexpression of
active beta-catenin/Wnt, in an embryonic chicken model, lead to an
enlarged liver with an expanded hepatocyte precursor cell population
[Suksaweang, S. et al., Dev Biol (2004) 266, 109-22]. BMP4, which was
shown to induce expression of numerous genes involved in Wnt signaling
[Nishanian, T. G. et al., Cancer Biol Ther (2004) 3, 667-75], was also up
regulated in Factor VIII KO SCID mice.

[0312] Transforming growth factor beta (TGF-beta), known to act as a
negative autocrine growth factor, was down regulated in the Factor VIII
KO SCID mice. Similarly, TGF-beta activated kinase 1 (TAK1) was
down-regulated in Factor VIII KO SCID mice. Thus, TGF-beta may contribute
to the increased organ size observed in hemophilic mice. TGF-beta and
TAK1 were previously shown to repress the expression of the telomerase
catalytic subunit (TERT) [Fujiki, T. et al., Oncogene (2007)]. The down
regulation of both TGF-beta and TAK1 in the factor VIII KO SCID mice was
therefore expected to result in increased expression of TERT, as was
indeed shown (data not shown).

[0316] In conclusion, inventors have shown in two different settings,
namely transplantation of embryonic precursor tissues as well as in G-CSF
induced splenomegaly, that Factor VIII, whose only known function is in
blood coagulation, also exhibits a novel role in organ size control via
regulation of genes.

Example 4

The Role of Coagulants Other than Factor VIII in Organ Size Control

[0317] Materials and Experimental Procedures

[0318] Animals

[0319] As described in detail in Example 2, hereinabove.

[0320] G-CSF Treatment

[0321] As described in detail in Example 2, hereinabove.

[0322] Clexane Treatment

[0323] Enoxaparine (Clexane 20 mg/0.2 ml, Rhone-poulenc, France) was used
at a dosage of 200 μg/mouse (dissolved in PBS) and 0.2 ml of the final
solution was injected subcutaneously into each mouse once a day.

[0337] As described hereinabove, the present findings indicated a role for
Factor VIII in organ size control. This intriguing role can either be
mediated directly by Factor VIII and/or through one of the other
coagulants activated along the cascade triggered by Factor VIII. As
illustrated in FIG. 7, Factor Xa is activated by Factor VIII and, in
turn, Thrombin is activated by Factor Xa. Thus, both factors could
potentially mediate the observed enhancement of organ size.

[0338] One approach to blocking Factor Xa and, to a lesser degree,
Thrombin is by the anti-coagulant Clexane, a low molecular weight heparin
derivative. Clexane binds to and accelerates the activity of
anti-thrombin III and thereby preferentially potentiates the inhibition
of Factors Xa and IIa (thrombin) (see FIG. 8A).

[0339] As can be seen in FIG. 8B, Clexane administration exhibited a
marked enhancement of the G-CSF induced splenomegaly. Indeed, the
contribution of Clexane treatment to splenomegaly was as effective as
that mediated by the Factor VIII KO mutation. Thus, following G-CSF
induction, the total average weight of the spleens was 266±91 mg in
C57BL/6 mice treated with Clexane compared to 261±154 mg in the Factor
VIII KO C57BL/6 mice (data not shown).

[0340] In parallel to G-CSF experiments, pig liver transplantation model
with and without Clexane administration was evaluated. As depicted in
FIG. 8C, implantation of pig embryonic liver affords a rapid assay as
growth can be monitored by the appearance of pig albumin (detectable by
specific ELISA) in the mouse serum as early as 7 days post transplant.
Importantly, Clexane administration in non-hemophilic recipients induced
marked enhancements of pig albumin blood levels on days 7 and 21 post
transplant compared to control recipients not receiving Clexane.

[0341] G-CSF Splenomegaly is Enhanced Upon Specific Inhibition of Thrombin
by Dabigatran

[0342] Considering that Clexane could potentially block not only factor Xa
but to some extent also Thrombin, further analysis was performed using a
more specific inhibitor of Thrombin, namely, Dabigatran. As can be seen
in FIGS. 9A-B, Dabigatran administration led to marked enhancement of the
G-CSF induced splenomegaly, similar to that exhibited by Clexane, and to
an enhancement of embryonic pig liver growth. These results mark thrombin
as a potential direct player in the enhanced splenomegaly phenotype (FIG.
9A) or in the enhancement of pig embryonic liver implants (FIG. 9B).
Thus, thrombin and anti-thrombin may be important candidates for further
manipulation of organ size.

[0344] Since thrombin could operate through Protease-Activated Receptor
(PAR) signaling and, especially, through a fine balance between the PAR1
and PAR4 receptors, inventors next evaluated the role of PAR signaling.
To this end, inventors tested PAR1 and PAR4 antagonists (as explained in
detail in the materials and experimental procedures section,
hereinabove). As can be seen in FIG. 10, daily treatment with the PAR1 or
PAR4 antagonist significantly enhanced G-CSF induced splenomegaly
(p<0.05).

Discussion

[0345] The models described above making use of heterologous embryonic
transplantation provided an additional tool in the study of growth
control, in that it enabled investigation of the cross-talk between
extrinsic host factors and intrinsic mechanisms relevant to size control
in the implant. In particular, this assay enabled pinpointing the role of
different extrinsic genes by using mutated or KO host mice. Thus,
inventors demonstrated, for the first time, that host Factor VIII plays a
critical role as an extrinsic factor in controlling the final size of
pig-derived organs.

[0346] The lack of any effect on mouse embryonic transplants, regardless
of whether the host or the donor was derived from a hemophilic or
non-hemophilic mouse, suggested that the role of Factor VIII is likely
limited to a growth checkpoint that only operates upon implantation of
tissue from a larger animal, such as the pig. Thus, assuming that mouse
and pig embryonic implants are endowed with stem cell pools of different
size prior to transplantation, they are likely to attain different organ
sizes upon completion of growth and differentiation in the mouse
recipients. Consequently, in hemophilic mice lacking the potential
inhibitory activity (i.e. overgrowth checkpoint) mediated by Factor VIII,
the size of the pig implants is likely to be larger, while mouse implants
growing to their normal expected size will not exhibit excessive growth,
and therefore will not be subject to Factor VIII control.

[0347] It could be argued that the role of Factor VIII might be limited to
the transplantation setting and thus might not have a true physiological
role in the intact animal. However, the results obtained in the context
of G-CSF induced splenomegaly, extended these findings to yet another
system in which a stimulus for overgrowth is also limited by factors of
the coagulation cascade.

[0348] Further interrogation of other factors downstream of Factor VIII in
the coagulation cascade revealed that thrombin is probably the Factor
VIII downstream factor that actually mediates regulation of oversize
control.

[0349] Thus, as outlined schematically in FIG. 11, interference with
steady state levels of factors of the coagulation cascade, as occurs in
Factor VIII KO mice or upon blockade of Factor Xa or thrombin, also
overcomes this checkpoint, thereby leading to oversized organs or to the
enhancement of G-CSF induced splenomegaly. The suggestion that thrombin
is a likely candidate for the actual inhibitory activity leading to
oversize control, is supported by its terminal position in the hemostatic
system, acting downstream of Factor VIII and Factor Xa. This hypothesis
was further supported by the demonstration that blockade of thrombin
receptors PAR1 or PAR4, similarly to thrombin blockade by Dabigatran, led
to enhancement of G-CSF induced splenomegaly.

[0350] In conclusion, the present data suggest a novel role for factors of
the coagulation cascade in organ size control. In particular, a role for
thrombin was pinpointed. This surprising finding may not only offer new
means to enhance or reduce the final size of implanted xenogeneic
embryonic tissues, but also adds a novel insight into the mysterious
question of organ size control in mammals.

[0351] Although the invention has been described in conjunction with
specific embodiments thereof, it is evident that many alternatives,
modifications and variations will be apparent to those skilled in the
art. Accordingly, it is intended to embrace all such alternatives,
modifications and variations that fall within the spirit and broad scope
of the appended claims.

[0352] All publications, patents and patent applications mentioned in this
specification are herein incorporated in their entirety by reference into
the specification, to the same extent as if each individual publication,
patent or patent application was specifically and individually indicated
to be incorporated herein by reference. In addition, citation or
identification of any reference in this application shall not be
construed as an admission that such reference is available as prior art
to the present invention. To the extent that section headings are used,
they should not be construed as necessarily limiting.